Phospholipid binding to the FAK catalytic domain impacts function.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• Focal adhesion kinase (FAK) is a crucial nonreceptor tyrosine kinase regulating cellular processes.
• FAK activation requires a conformational shift from an autoinhibited to an active state.
• Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) binding to the FERM domain is known to release FAK autoinhibition.

Purpose of the Study:

• To investigate the role of the FAK catalytic domain in PI(4,5)P2 binding and activation.
• To validate in silico predictions of PI(4,5)P2 interaction sites within the FAK catalytic domain.
• To assess the functional significance of catalytic domain-mediated phospholipid binding in FAK's biochemical and biological activities.

Main Methods:

• Engineering mutations in FAK to disrupt putative PI(4,5)P2 binding sites in the catalytic domain.
• Assessing FAK mutant phosphorylation levels and their ability to rescue fak-/- fibroblasts.
• Performing in vitro binding assays to measure PI(4,5)P2 and other phosphoinositide binding to wild-type and mutant FAK catalytic domains.

Main Results:

• Mutant FAK proteins with disrupted catalytic domain PI(4,5)P2 binding sites showed impaired phosphorylation and incomplete rescue of fak-/- fibroblast phenotypes.
• The FAK catalytic domain demonstrated in vitro binding to PI(4,5)P2, which was abolished by mutations.
• Catalytic domain binding was not selective for PI(4,5)P2, showing affinity for various phosphoinositides. The most defective mutant showed reduced phosphoinositide binding.

Conclusions:

• The FAK catalytic domain directly binds to phosphoinositides, including PI(4,5)P2.
• Specific residues in the catalytic domain are important for PI(4,5)P2 binding and FAK activation.
• Catalytic domain phospholipid binding is essential for both the biochemical activity and biological function of FAK.